Androgen-dependent Prostate Cancer Cells Research Articles

Abstract 3223: Embryonic ectoderm development (EED) inhibitor APG-5918 (EEDi-5273) and MDM2 inhibitor alrizomadlin (APG-115) synergistically inhibit tumor growth in preclinical models of prostate cancer (PCa)

Abstract Advanced PCa, especially castration-resistant PCa, represents an urgent unmet treatment need. Dysregulation of the polycomb repressive complex 2 (PRC2), which mediates trimethylation of histone H3 at lysine 27 (H3K27me3) and leads to gene silencing, is common in PCa and correlates with poor prognosis. EED, a core PRC2 component, is crucial for histone methyltransferase activity. MDM2, a negative regulator of p53, is often amplified or overexpressed in PCa and associated with metastasis and poor clinical outcomes. We evaluated antitumor activity and molecular mechanisms of EED inhibitor APG-5918/EEDi-5273 and MDM2 selective inhibitor alrizomadlin in preclinical PCa models. Human castration-resistant (22Rv1) and androgen-dependent (LNCaP) PCa cell lines were used in antiproliferation assays. Cell viability was determined by CellTiter-Glo luminescent assays. Western blotting was used to explore mechanisms of action. Subcutaneous 22Rv1 and LNCaP cell line-derived xenograft (CDX) models were established to evaluate antitumor effects of APG-5918 and alrizomadlin in vivo. Single-agent APG-5918 or alrizomadlin had limited inhibition on PCa cell growth; combined, they demonstrated synergistic antitumor activity. In the 22Rv1 CDX model, single-agent alrizomadlin (100 mg/kg) or APG-5918 (100 mg/kg) administered for 26 days exerted no/limited antitumor activity, with treatment-to-control (T/C) values of 93.39% and 49.89%, respectively. In contrast, the doublet enhanced antitumor activity (T/C value, 33.22%), with a synergistic index of 1.40. In the LNCaP CDX model, APG-5918 (100 mg/kg) administered for 28 days exhibited minimal antitumor effects (T/C value on last day of dosing, 80.32%). Alrizomadlin (100 or 50 mg/kg) showed limited antitumor activity (T/C value, 47.61%). Combined treatment significantly enhanced antitumor activity (T/C value, 15.69%), with a synergistic index of 2.44. Regarding putative mechanisms, western blot analyses showed that APG-5918 downregulated oncogenic drivers and DNA methylation factors (UHRF1, DNMT1) and on-target-related pharmacodynamic markers (H3K27me3, EED, EZH2). Alrizomadlin markedly downregulated UHRF1 and DNMT1, and upregulated p53 and p21 expression. Combined treatment further enhanced downregulation of DNMT1, UHRF1, cell cycle pathway proteins (pRb, CDK6), antiapoptotic protein MCL-1, and synergistically increased cleavage of PARP-1, a marker of apoptosis. In summary, we show that targeting EED and MDM2 synergistically enhances antitumor activity in PCa cell lines and CDX models. Mechanistically, this combination likely modulated pathways related to DNA methylation, cell cycle, and apoptosis. The findings provide a scientific rationale for future clinical development of APG-5918 and alrizomadlin for PCa patients. Citation Format: Yan Yin, Yun Yang, Eric Liang, Baisong Li, Hengbang Wang, Dajun Yang, Yifan Zhai. Embryonic ectoderm development (EED) inhibitor APG-5918 (EEDi-5273) and MDM2 inhibitor alrizomadlin (APG-115) synergistically inhibit tumor growth in preclinical models of prostate cancer (PCa) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3223.

Abstract 5695: miR-5787 suppresses cancer cell growth as a Fe3O4-nanoparticles induced pivotal miRNA

Abstract Nanotechnology offers great potential for cancer diagnosis and treatments. Especially, nanoparticles (NPs) have been examined and utilized as drug delivery systems, hyperthermia and combination therapy. We have examined and reported the combined effects of Fe3O4-nanoparticles (Fe3O4-NPs) with chemotherapeutic agents on prostate cancer cell in vitro. miRNAs are an important factor in cancer progression, and elucidating the miRNA-mediated intracellular network of cancer cells could suggest new cancer treatment strategies. In this study, to identify microRNAs (miRNAs) associated with anticancer effects of Fe3O4-NPs, the comprehensive expression of miRNAs by Microarray (Agilent G4780A SuperPrint G3 Human v16 miRNA 8x60K Microarray kit) in A549 (lung cancer cell line), LNCaP (androgen-dependent prostate cancer cell line) and DU145 (androgen-independent prostate cancer cell line) under Fe3O4-NPs exposure was performed. miR-5787 was one of the increased miRNAs in three cancer cell lines under Fe3O4-NPs, and its increase in miR-5787 was due to the reactive oxygen species (ROS) production by Fe3O4-NPs. The additional analysis suggested that miR-5787 might suppressed the cancer cell growth by targeting the eIF5. In this study, we identified miR5787 as a Fe3O4-NPs induced pivotal miRNA in cancer cells, and can be used as a therapeutic target. Citation Format: Masatoshi Watanabe, Hitoshi Nakano, Chise Matsuda, Eri Usugi, Hiroshi Imai, Yoshifumi Hirokawa, Kazutoshi Iijima. miR-5787 suppresses cancer cell growth as a Fe3O4-nanoparticles induced pivotal miRNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5695.

Anticancer Effect of Theranekron® on Androgen-Dependent Prostate Cancer Cells.

Prostate cancer (PCa) is a significant health problem in men worldwide. Although there are numerous treatment choices for PCa, acquired resistance limits treatment success. Therefore, there is a need for new approaches as powerful resources for use in alternative or supportive therapeutic strategies for anticancer therapeutics. Theranekron® is a commercially available alcoholic extract of Tarantula cubensis. Recent studies have shown the potent anticancer effect of theranekron in human tumors, including PCa. Herein, we comparatively examined the antiproliferative activity of theranekron and its biochemical action on androgenic signaling and cell cycle-related cyclin proteins in androgen-dependent PCa cells, LNCaP, VCaP, and 22Rv1. Human androgen-dependent PCa cells, LNCaP (CRL-1740TM), 22Rv1 (CRL-2505TM), and VCaP (CRL-2876TM) were used to evaluate the effect of theranekron in vitro. The impact of theranekron on cell viability was evaluated using a WST-1-based viability test. Its impact on AR, cyclin A2, cyclin B1, and cyclin E1 was examined by immunoblotting. To test the anti-malignant effect of theranekron on 3D tumor formation of PCa cells, soft agar assay was used. Our results indicated that theranekron treatment significantly reduced the viability of PCa cells. It remarkably decreased the protein levels of AR, cyclin A2, cyclin B1, and cyclin E1 in a dose-dependent manner. In addition, Theranekron administration strongly limited the 3D tumor formation of LNCaP, 22Rv1, and VCaP cells. Our findings strongly suggest that theranekron may offer potent therapeutic efficacy against androgen-dependent PCa cells. Moreover, it may be a potent component for preventing acquired resistance to chemotherapeutics.

Analisis Dinamik Model Sel Kanker Prostat dengan Terapi Vaksin Kuratif

Prostate cancer is a type of cancer that occurs in men and requires an effective therapeutic approach. Treatment of prostate cancer depends on the stage at diagnosis. In advanced stages of prostate cancer can be treated with hormone therapy such as chemotherapy which is then followed by vaccine therapy which aims to help increase the body's immune system response to prostate cancer cells. This model consists of a system of ordinary differential equations with five variables used, including androgen-dependent prostate cancer cells, androgen-independent prostate cancer cells, dendritic cells, effector cells, and curative vaccines. Then two equilibrium point conditions are produced, when there is no vaccine for disease free conditions and endemic conditions , then when the vaccine there are three equilibrium conditions namely disease free , side effects and local existence between prostate cancer cells with vaccine . The results of the stability analysis for each equilibrium point show that when , the condition is global asymptotic, while the condition is stable because the eigenvalue is negative. When for the condition it is unstable because the two roots are positive, then for the condition it is global asymptotic and for the condition it is asymptotically local because all the eigenvalues are negative. The numerical simulations of equilibrium points obtained using the fourth order runge-kutta method according to different q parameter values show that the larger the dendritic cells and effector cells activated, the greater the vaccine that enters the body, resulting in immune cells that will fight prostate cancer cells.

New mode of action of curcumin on prostate cancer cells: Modulation of endoplasmic reticulum-associated degradation mechanism and estrogenic signaling.

Prostate cancer is leading to cancer-related mortality in numerous men each year worldwide. While there are several treatment options, acquired drug resistance mostly limits the success of treatments. Therefore, there is a need for the development of innovative treatments. Curcumin is one of the bioactive polyphenolic ingredients identified in turmeric and has numerous biological activities, such as anti-inflammatory and anticancer. In the present study, we investigated the effect of curcumin on the ER-associated degradation (ERAD) and estrogenic signaling in prostate cancer cells. The antiproliferative effect of curcumin on human androgen-dependent prostate cancer cell lines LNCaP and VCaP was estimated by WST-1 assay. Morphological alterations were investigated with an inverted microscope. We investigated the effect of curcumin on ERAD and estrogen signaling proteins by immunoblotting assay. To evaluate the impact of curcumin on endoplasmic reticulum (ER) protein quality-related, the expression level of 32 genes was analyzed by quantitative reverse transcription polymerase chain reaction. The nuclear translocation of estrogen receptor was examined by nuclear fractionation and immunofluorescence microscopy. We found that curcumin effectively reduced the proliferation rates of LNCaP and VCaP cells. ERAD proteins; Hrd1, gp78, p97/VCP, Ufd1 and Npl4 were strongly induced by curcumin. Also, the steady-state level of polyubiquitin was increased in a dose-dependent manner in both cell lines. Curcumin administration remarkably decreased the protein levels of estrogen receptor-alfa (Erα), whereas estrogen receptor-beta unaffected. Additionally, curcumin strongly restricted the nuclear translocation of Erα. Present data suggest that curcumin may be effectively used in therapeutic approaches associated with the targeting ER protein quality control mechanism and modulation of estrogen signaling in prostate cancer.

Integrative analysis of transcriptomic and metabolomic profiles reveals enhanced arginine metabolism in androgen-independent prostate cancer cells

BackgroundProstate cancer is a common solid tumor that affects a significant number of men worldwide. Conventional androgen deprivation therapy (ADT) increases the risk of developing castration-resistant prostate cancer (CRPC). Effective clinical management of patients with CRPC is challenging due to the limited understanding.MethodsIn this study, transcriptomic and metabolomic profiles of androgen-dependent prostate cancer cell line LNCaP and the androgen-independent cells developed from LNCaP cells (LNCaP-ADR) were investigated using RNA-sequencing and LC-MS/MS, respectively. The differentially expressed genes and metabolites were analyzed, and integrative analysis of transcriptomic and metabolomic data was further conducted to obtain a comprehensive understanding of the metabolic characteristics in LNCaP-ADR cells. Quantitative real-time PCR (QPCR) was employed to ascertain the mRNA expression levels of the selected differentially expressed genes.ResultsThe arginine and proline metabolism pathway was identified as a commonly altered pathway at both the transcriptional and metabolic levels. In the LNCaP-ADR cells, significant upregulation was observed for metabolites including 5-Aminopentanoic acid, L-Arginine, L-Glutamic acid, N-Acetyl-L-alanine, and Pyrrole-2-carboxylic acid at the metabolic level. At the transcriptional level, MAOA, ALDH3A2, ALDH2, ARG1, CKMT2, and CNDP1 were found to be significantly upregulated in the LNCaP-ADR cells. Gene set enrichment analysis (GSEA) identified various enriched gene sets in the LNCaP-ADR cells, encompassing inflammatory response, 9plus2 motile cilium, motile cilium, ciliary plasm, cilium or flagellum-dependent cell motility, cilium movement, cilium, response to endoplasmic reticulum stress, PTEN DN.V1 DN, SRC UP.V1 UP, IL15 UP.V1 DN, RB DN.V1 DN, AKT UP MTOR DN.V1 UP, VEGF A UP.V1 UP, and KRAS.LUNG.BREAST UP.V1 UP.ConclusionsThese findings highlight the substantial association between the arginine and proline metabolism pathway and CRPC, emphasizing the need to prioritize strategies that target dysregulated metabolites and differentially expressed genes as essential interventions in the clinical management of CRPC.

Antiproliferative, apoptosis-inducing, and GSTP1 demethylation activities of Ellagitannins isolated from Cornus alba L.

This study aimed to prove the prostate cancer chemopreventive activity of compounds isolated from CA. We evaluated these compounds using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and evaluated their NF-κB inhibitory activity and apoptosis-inducing activity using western blot analysis and flow cytometry, respectively. Their DNA methylation activity was also evaluated via a methylation-specific polymerase chain reaction in androgen-dependent (LNCaP) and androgen-independent (PC-3) prostate cancer cell lines. Camptothin B (1), cornusiin B (2), and cornusiin A (3), which were isolated in our previous work, relatively reduced the protein expression levels in PCa cells. Among them, cornusiin B (2) exhibited excellent NF-κB inhibitory activity. Also, concentration-dependently increased the unmethylated DNA content and decreased the methylated DNA content in both PC-3 and LNCaP cells. Therefore, cornusiin B (2), which was isolated from CA, has the potential to act as a chemopreventive agent for prostate cancer.

Ratiometric Inclusion of Fibroblasts Promotes Both Castration-Resistant and Androgen-Dependent Tumorigenic Progression in Engineered Prostate Cancer Tissues.

To investigate the ratiometric role of fibroblasts in prostate cancer (PCa) progression, this work establishes a matrix-inclusive, 3D engineered prostate cancer tissue (EPCaT) model that enables direct coculture of neuroendocrine-variant castration-resistant (CPRC-ne) or androgen-dependent (ADPC) PCa cells with tumor-supporting stromal cell types. Results show that the inclusion of fibroblasts within CRPC-ne and ADPC EPCaTs drives PCa aggression through significant matrix remodeling and increased proliferative cell populations. Interestingly, this is observed to a much greater degree in EPCaTs formed with a small number of fibroblasts relative to the number of PCa cells. Fibroblast coculture also results in ADPC behavior more similar to the aggressive CRPC-ne condition, suggesting fibroblasts play a role in elevating PCa disease state and may contribute to the ADPC to CRPC-ne switch. Bulk transcriptomic analyses additionally elucidate fibroblast-driven enrichment of hallmark gene sets associated with tumorigenic progression. Finally, the EPCaT model clinical relevancy is probed through a comparison to the Cancer Genome Atlas (TCGA) PCa patient cohort; notably, similar gene set enrichment is observed between EPCaT models and the patient primary tumor transcriptome. Taken together, study results demonstrate the potential of the EPCaT model to serve as a PCa-mimetic tool in future therapeutic development efforts.

Canonical and Nuclear mTOR Specify Distinct Transcriptional Programs in Androgen-Dependent Prostate Cancer Cells.

The finding that canonical and nuclear mTOR signaling pathways control distinct gene programs opens therapeutic opportunities to modulate mTOR activity in prostate cancer cells.

Automation, live-cell imaging, and endpoint cell viability for prostate cancer drug screens.

Androgen deprivation therapy (ADT) is the standard of care for high risk and advanced prostate cancer; however, disease progression from androgen-dependent prostate cancer (ADPC) to lethal and incurable castration-resistant prostate cancer (CRPC) and (in a substantial minority of cases) neuroendocrine prostate cancer (NEPC) is common. Identifying effective targeted therapies is challenging because of acquired resistance to established treatments and the vast heterogeneity of advanced prostate cancer (PC). To streamline the identification of potentially active prostate cancer therapeutics, we have developed an adaptable semi-automated protocol which optimizes cell growth and leverages automation to enhance robustness, reproducibility, and throughput while integrating live-cell imaging and endpoint viability assays to assess drug efficacy in vitro. In this study, culture conditions for 72-hr drug screens in 96-well plates were established for a large, representative panel of human prostate cell lines including: BPH-1 and RWPE-1 (non-tumorigenic), LNCaP and VCaP (ADPC), C4-2B and 22Rv1 (CRPC), DU 145 and PC3 (androgen receptor-null CRPC), and NCI-H660 (NEPC). The cell growth and 72-hr confluence for each cell line was optimized for real-time imaging and endpoint viability assays prior to screening for novel or repurposed drugs as proof of protocol validity. We demonstrated effectiveness and reliability of this pipeline through validation of the established finding that the first-in-class BET and CBP/p300 dual inhibitor EP-31670 is an effective compound in reducing ADPC and CRPC cell growth. In addition, we found that insulin-like growth factor-1 receptor (IGF-1R) inhibitor linsitinib is a potential pharmacological agent against highly lethal and drug-resistant NEPC NCI-H660 cells. This protocol can be employed across other cancer types and represents an adaptable strategy to optimize assay-specific cell growth conditions and simultaneously assess drug efficacy across multiple cell lines.

Castration promotes the browning of the prostate tumor microenvironment

BackgroundAdipose tissue has gained attention due to its potential paracrine role. Periprostatic adipose tissue surrounds the prostate and the prostatic urethra, and it is an essential player in prostate cancer progression. Since obesity is directly related to human tumor progression, and adipose tissue depots are one of the significant components of the tumor microenvironment, the molecular mediators of the communication between adipocytes and epithelial cells are in the spotlight. Although periprostatic white adipose tissue contributes to prostate cancer progression, brown adipose tissue (BAT), which has beneficial effects in metabolic pathologies, has been scarcely investigated concerning cancer progression. Given that adipose tissue is a target of androgen signaling, the actual role of androgen removal on the periprostatic adipose tissue was the aim of this work.MethodsSurgical castration of the transgenic adenocarcinoma of the mouse prostate (TRAMP) was employed. By histology examination and software analysis, WAT and BAT tissue was quantified. 3T3-like adipocytes were used to study the role of Casodex® in modifying adipocyte differentiation and to investigate the function of the secretome of adipocytes on the proliferation of androgen-dependent and independent prostate cancer cells. Finally, the role of cell communication was assayed by TRAMP-C1 xenograft implanted in the presence of 3T3-like adipocytes.ResultsAndrogen removal increases brown/beige adipose tissue in the fat immediately surrounding the prostate glands of TRAMP mice, concomitant with an adjustment of the metabolism. Castration increases body temperature, respiratory exchange rate, and energy expenditure. Also, in vitro, it is described that blocking androgen signaling by Casodex® increases the uncoupling protein 1 (UCP1) marker in 3T3-like adipocytes. Finally, the effect of brown/beige adipocyte secretome was studied on the proliferation of prostate cancer cells in vivo and in vitro. The secretome of brown/beige adipocytes reduces the proliferation of prostate cancer cells mediated partly by the secretion of extracellular vesicles.ConclusionsConsequently, we concluded that hampering androgen signaling plays a crucial role in the browning of the periprostatic adipose tissue. Also, the presence of brown adipocytes exhibits the opposite effect to that of white adipocytes in vitro regulating processes that govern the mechanisms of cell proliferation of prostate cancer cells. And finally, promoting the browning of adipose tissue in the periprostatic adipose tissue might be a way to handle prostate cancer cell progression.EECkevEGf-HMwkJP3j4bZsVideo

Neferine Targets the Oncogenic Characteristics of Androgen-Dependent Prostate Cancer Cells via Inducing Reactive Oxygen Species.

Castration resistance poses a significant challenge in the management of advanced prostate cancer (PCa), with androgen deprivation therapy (ADT) or chemotherapy being the primary treatment options. However, these approaches often lead to significant side effects and the development of therapeutic resistance. Therefore, it is crucial to explore novel treatment options that can efficiently target PCa, improve patient survival, and enhance their quality of life. Neferine (Nef), a bioactive compound derived from plants, has emerged as a promising candidate for cancer treatment due to its ability to induce apoptosis, autophagy, and cell cycle arrest. In this study, we investigated the potential anticancer effects of Nef in androgen receptor (AR)-positive LNCaP and VCaP cells, representative models of androgen-dependent PCa. Our findings demonstrate that Nef effectively inhibits cell growth, proliferation, and the tumorigenic potential of androgen-dependent PCa cells. Furthermore, Nef treatment resulted in the excessive production of reactive oxygen species (ROS), leading to the activation of key markers of autophagy and apoptosis. These results suggest that Nef has the potential to target the oncogenic characteristics of androgen-dependent PCa cells by exploiting the potency of ROS and inducing autophagy and apoptosis in AR-positive PCa cells. These findings shed light on the therapeutic potential of Nef as a novel treatment option with reduced side effects for androgen-dependent prostate cancer. Further investigations are warranted to assess its efficacy and safety in preclinical and clinical settings.

F77 antigen is a promising target for adoptive T cell therapy of prostate cancer

Adoptive immunotherapy using chimeric antigen receptor (CAR) T cells has made significant success in treating hematological malignancies, paving the way for solid tumors like prostate cancer. However, progress is impeded by a paucity of suitable target antigens. A novel carbohydrate antigen, F77, is expressed on both androgen-dependent and androgen-independent prostate cancer cells, making it a potential immunotherapy target. This study entails the generation and evaluation of a second-generation CAR against a carbohydrate antigen on malignant prostate cancer cells. Using a single chain fragment variable (scFv) from an F77-specific mouse monoclonal antibody, we created second-generation CARs with CD28 and CD137 (4-1BB) costimulatory signals. F77 expressing lentiviral CAR T cells produce cytokines and kill tumor cells in a F77 expression-dependent manner. These F77-specific CAR T cells eradicate prostate tumors in a human xenograft model employing PC3 cells. These findings validate F77 as a promising immunotherapeutic target for prostate cancer and other malignancies with this aberrant carbohydrate structure.

Investigation of the Effect of Myricetin On Human Androgen Dependent Prostate Cancer Cells

Prostat kanseri dünyada erkekler arasında en sık görülen ikinci kanser türüdür. Prostat kanserinin morbidite ve mortalitesi son zamanlarda artmıştır. Tedavisi için birçok alternatif yaklaşımlar geliştirilmeye çalışılsa da, prostat kanseri hala kötü prognoz sergilemekte ve yüksek ölüm oranları ile karşılaşılmaktadır. Myricetin, antikanser özelliği ile ilgi çeken doğal bir flavonoid bileşiktir. Yapılan in vitro ve in vivo çalışmalar myricetinin çeşitli mekanizmalar yoluyla prostat kanserini etkili bir şekilde inhibe ettiğini göstermektedir. Bu çalışmanın amacı artan dozlarda myricetin uygulamasının androjen reseptör bağımlı insan prostat kanser hücre hattı olan LNCaP hücrelerinin canlılığı üzerindeki etkilerini belirlemek ve apoptozla ilişkili BAX ve BCL2 genlerinin ekspresyon seviyelerini tespit etmektir. LNCaP hücreleri myricetinin 10 μM, 25 μM, 50 μM, 100 μM, 150 μM’lık konsantrasyonları ile 24 ve 48 saat süresince inkübe edilmiş ve hücre canlılığındaki değişimler 2,3-bis-(2-metoksi-4-nitro-5-sulfofenil)-2Htetrazolyum-5-karboksanilid (XTT) yöntemiyle belirlenerek IC50 değerleri hesaplanmıştır. BAX ve BCL2 gen ifadelerindeki değişimler ise Real-Time PCR metoduyla belirlenmiş ve elde edilen verilerin analizinde ΔΔCT metodu kullanılmıştır. Myricetinin uygulanan bütün dozlarda kontrole göre LNCaP hücre canlılığını azalttığı gösterilmiş olup IC50 değeri 24. saat için 123.76 μM, 48. saat için ise 84.79 μM olarak tespit edilmiştir. Ayrıca, myricetin uygulamasının apoptoz ilişkili BAX gen ifadesini istatistiksel olarak anlamlı bir düzeyde artırırken BCL2 gen ifadesini ise azalttığı görülmüştür. Myricetinin LNCaP hücrelerindeki antiproliferatif ve apoptotik etkileri daha detaylı olarak araştırılmalıdır.

Triiodothyronine positively regulates endoplasmic reticulum-associated degradation (ERAD) and promotes androgenic signaling in androgen-dependent prostate cancer cells

Thyroid hormones (THs) play crucial roles in numerous physiological processes of nearly all mammalian tissues, including differentiation and metabolism. Deterioration of TH signaling has been associated with several pathologies, including cancer. The effect of highly active triiodothyronine (T3) has been investigated in many in vivo and in vitro cancer models. However, the role of T3 on cancerous prostate tissue is controversial today. Recent studies have focused on the characterization of the supportive roles of the endoplasmic reticulum-associated degradation (ERAD) and unfolded protein response (UPR) signaling in prostate cancer (PCa) and investigating new hormonal regulation patterns, including estrogen, progesterone and 1,25(OH)2D3. Additionally, androgenic signaling controlled by androgens, which are critical in PCa progression, has been shown to be regulated by other steroid hormones. Today, the effects of T3 on ERAD and UPR are unknown, the impact on androgenic signaling is also still not fully understood in PCa. Therefore, we aimed to investigate the molecular action of T3 on the ERAD mechanism and UPR signaling in PCa cells and also extensively examined the effect of T3 on androgenic signaling. Our data indicated that T3 tightly regulated ERAD and UPR signaling in androgen-dependent PCa cells. We also found that T3 hormone stimulated androgenic signaling by upregulating AR mRNA and protein levels and enhancing its nuclear translocation. Additionally, advanced computational studies supported the ligand binding effect of T3 on AR protein. Our data suggest that targeting thyroidal signaling should be considered in therapeutic approaches to be developed for prostate malignancy in addition to other steroidal regulations.

Abstract B008: Tumor-stromal 3D co-cultures to study the role of cancer-associated fibroblasts in the acquisition of androgen-deprivation therapy resistance in prostate cancer

Abstract Introduction and Objective Several studies have shown that cancer-associated fibroblasts (CAFs), the most abundant component in prostate cancer (PCa) microenvironment, promote resistance to androgen deprivation therapies, and metastatic development. The aim of this study is to elucidate the mechanisms of CAF-induced therapy resistance through the establishment of in vitro 3D co-cultures. Methods In this study, the androgen-dependent PNPCa patient-derived xenograft (PDX) model (derived from soft tissue metastasis), and the androgen-independent LAPC9 PDX model (bone metastasis) are used. Epithelial and stromal cells (human and mouse origin, respectively) are separated through magnetic-associated cell sorting to generate PDX-derived organoids and fibroblasts. Fibroblast RNA and protein expression is assessed through RT-qPCR, Western Blot, and Immunofluorescence. Direct 3D co-cultures are obtained by combining fluorescently labelled organoids and fibroblasts in ultra-low attachment plates in defined media. In indirect co-cultures, organoids are cultured in Spherical Plates (Kugelmeiers) with fibroblasts on transwell inserts. Organoid viability is measured after 9 days by CellTiter-Glo 3D Assay. Results PDX-derived fibroblasts express typical CAF genes (e.g. α-smooth muscle actin and Tenascin C), and androgen receptor (AR), both at RNA and protein level. AR protein expression (as well as RNA expression of the AR target gene Fkbp5) is increased in CAFs upon treatment with dyhydrotestosterone (DHT) 10nM, while co-treatment with the AR inhibitor Enzalutamide 10mM abrogates this effect, suggestive of functional AR signaling. These CAFs can organize into tumor/CAF organoid 3D structures upon combination with PDX-derived tumor epithelial cells, providing an in vitro functional model to study tumor-stromal interactions. In indirect (transwell) co-cultures, CAFs increase the viability of the androgen dependent PNPCa organoids, but not that of androgen independent LAPC9. This growth-promoting effect is observed even upon culturing of PNPCa organoids in non-optimal growth conditions, at low DHT concentrations (DHT 0.5nM and 0.25nM versus the standard DHT 1nM). The factors mediating this process will be identified by gene expression profiling of CAFs and organoids in transwell co-cultures and/or monoculture. Conclusions Our data indicate that PCa metastatic cells modify the properties of neighbor stromal cells to support tumorigenesis, since PDX-derived fibroblasts, originally part of the mouse dermal stroma, display CAF features and active AR signaling. 3D co-cultures represent a useful tool to study the tumor-stromal interactions. Our transwell co-culture experiments suggest that CAFs play an important role in supporting the viability and growth of early metastatic androgen dependent PCa cells (PNPCa) by secreting pro-tumorigenic factors. The possibility that CAFs exert different effects depending on the tumor stage setting (early vs advanced) will be subsequently investigated. Citation Format: Francesco Bonollo, Natalie Sampson, George Thalmann, Sofia Karkampouna, Marianna Kruithof-de Julio. Tumor-stromal 3D co-cultures to study the role of cancer-associated fibroblasts in the acquisition of androgen-deprivation therapy resistance in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B008.

Clostridium scindens metabolites trigger prostate cancer progression through androgen receptor signaling.

Prostate cancer (PCa) is one of the most common malignancies in men; recently, PCa-related mortality has increased worldwide. Although androgen deprivation therapy (ADT) is the standard treatment for PCa, patients often develop aggressive castration-resistant PCa (CRPC), indicating the presence of an alternative source of androgen. Clostridium scindens is a member of the gut microbiota and can convert cortisol to 11β-hydroxyandrostenedione (11β-OHA), which is a potent androgen precursor. However, the effect of C. scindens on PCa progression has not been determined. In this study, androgen-dependent PCa cells (LNCaP) were employed to investigate whether C. scindens-derived metabolites activate androgen receptor (AR), which is a pivotal step in the development of PCa. Results showed that cortisol metabolites derived from C. scindens-conditioned medium promoted proliferation and enhanced migration of PCa cells. Furthermore, cells treated with these metabolites presented activated AR and stimulated AR-regulated genes. These findings reveal that C. scindens has the potential to promote PCa progression via the activation of AR signaling. Further studies on the gut-prostate axis may help unravel an alternative source of androgen that triggers CRPC exacerbation.

Delivery of curcumin within emulsome nanoparticles enhances the anti-cancer activity in androgen-dependent prostate cancer cell.

Curcumin, a dietary polyphenol isolated from turmeric, is a potent phytochemical possessing intrinsic anticancer activities against various cancer types including prostate cancer. However, low water solubility and bioavailability of the compound are major challenges against its medical use. The objective of this study is to evaluate the therapeutic potential of curcumin-loaded emulsome nanoparticular system, i.e. CurcuEmulsomes, for the treatment of androgen dependent LNCaP prostate cancer cell line. The antiproliferative effect of both free curcumin and CurcuEmulsome were investigated comparatively on LNCaP and PNT1A cells. Cell viability data indicates that the inhibition in proliferation of LNCaP cells becomes more effective when curcumin is provided with its emulsome formulation rather than its free form. Corresponding to a therapeutic index of 2.25, Half maximal inhibitory (IC50) and cytotoxic (CC50) concentrations of CurcuEmulsomes for LNCaP and PNT1A cells were estimated as 17.1µM and 38.6µM, respectively. The fluorescence signal of autofluorescence curcumin was preserved within the CurcuEmulsomes at72h after the treatment. Thus, CurcuEmulsomes prolonged biological activity of curcumin. Induced apoptotic cell death and stimulated cell cycle arrest at G2/M phase were attributed to antiproliferative activity of CurcuEmulsomes. Treatment of LNCaP cells with CurcuEmulsomes increased expression of caspase-3 significantly by 11.76-fold, whereas decreased cyclin D1, Bcl-2 and AR expression levels significantly by of 0.18, 0.06 and 0.46-fold, respectively. Presented safety and anticancer activity of CurcuEmulsomes on LNCaP cell line highlights the potential of CurcuEmulsomes to benefit intrinsic anticancer activities of curcumin in androgen dependent prostate cancer therapy.

Dynamic phase separation of the androgen receptor and its coactivators key to regulate gene expression.

Numerous cancers, including prostate cancer (PCa), are addicted to transcription programs driven by specific genomic regions known as super-enhancers (SEs). The robust transcription of genes at such SEs is enabled by the formation of phase-separated condensates by transcription factors and coactivators with intrinsically disordered regions. The androgen receptor (AR), the main oncogenic driver in PCa, contains large disordered regions and is co-recruited with the transcriptional coactivator mediator complex subunit 1 (MED1) to SEs in androgen-dependent PCa cells, thereby promoting oncogenic transcriptional programs. In this work, we reveal that full-length AR forms foci with liquid-like properties in different PCa models. We demonstrate that foci formation correlates with AR transcriptional activity, as this activity can be modulated by changing cellular foci content chemically or by silencing MED1. AR ability to phase separate was also validated in vitro by using recombinant full-length AR protein. We also demonstrate that AR antagonists, which suppress transcriptional activity by targeting key regions for homotypic or heterotypic interactions of this receptor, hinder foci formation in PCa cells and phase separation in vitro. Our results suggest that enhanced compartmentalization of AR and coactivators may play an important role in the activation of oncogenic transcription programs in androgen-dependent PCa.

Prostate cancer small extracellular vesicles participate in androgen-independent transformation of prostate cancer by transferring let-7a-5p

ObjectivesAndrogen deprivation therapy (ADT) is a standard treatment for advanced prostate cancer (PCa). However, after 2–3 years ADT treatment, prostate cancer inevitably transits from androgen-dependent PCa (ADPC) to androgen-independent PCa (AIPC), which has a poor prognosis owing to its unclear mechanism and lack of effective therapeutic targets. Small extracellular vesicles (sEVs) play a vital role in the development of cancer. However, the role of PCa sEVs in the transformation of AIPC remains poorly understood. Materials and methodsTwo different cell models were employed and compared. sEVs from ADPC cells (LNCaP) and AIPC cells (LNCaP-AI + F cells) were isolated and characterized. After co-culture of LNCaP-AI + F sEVs with LNCaP cells and of LNCaP sEVs with LNCaP-AI + F cells, androgen-independent transformation was determined respectively. Mechanically, small RNA sequencing was performed. Androgen-independent transformation was examined by the upregulation and downregulation of miRNA and downstream pathways were analyzed. ResultsLNCaP-AI + F sEVs promoted the androgen-independent transformation of LNCaP cells. Interestingly, LNCaP sEVs exhibited a capacity to reverse the process.Let-7a-5p transfer was demonstrated. Furthermore, let-7a-5p overexpression promotes the androgen-independent transformation and let-7a-5p down-regulation reverses the process. Androgen receptor (AR) and PI3K/Akt pathways were identified and demonstrated by both let-7a-5p regulation and PCa sEVs coculture. ConclusionsPCa sEVs are intimately involved in the regulation of androgen-independent transformation of prostate cancer by transferring the key sEVs molecular let-7a-5p and then activating the AR and PI3K/Akt signaling pathways. Our results provide new perspectives for the development of sEVs and sEVs molecular targeted treatment approaches for AIPC patients.